Human rhinovirus (HRV) infections are among the most frequent cause of the common cold. (Pitkaranta, A., and F. G. Hayden. 1998. Ann. Med. 30:529-537). Recently HRVs have been linked to severe lower respiratory illnesses in young children (Miller E. K., 2007. J. Infect. Dis. 195:773-781, Monto A. S., Clin. Ther. 2001; 23:1615-1627), the elderly (Hicks, L. A., J. Am. Geriatr. Soc., 2006; 54:284-289, Nicholson, K. G., Br. Med. J., 1996; 313:1119-1123, Wald, T., Ann. Intern. Med., 1995; 123:588-593) and the immunocompromised (Gosh, S. R., Clin. Infect. Dis., 1999; 29:528-532, Ison, M. G., Clin. Infect. Dis., 2003; 36:1139-1143). Persons with underlying respiratory disease, like asthma, chronic bronchitis and cystic fibrosis may also have increased risk of severe HRV-associated complications. (Friedlander, S. L., and W. W. Busse, J. Allergy. Clin. Immunol., 2005; 116:267-273; Khetsuriani, N, J. Allergy. Clin. Immunol., 2007; 119:314-321; Smyth, A. R., Arch. Dis. Child., 1995; 73:117-120).
The family Picornaviridae contains HRVs together with the human enteroviruses (HEVs) (King, A. M., et al. 2000. Picornaviridae, p. 657-678. In Virus Taxonomy. Seventh Report of the International Committee for the Taxonomy of Viruses. Academic Press, San Diego, Calif.). At least 100 distinct HRV serotypes of this family are assigned to two phylogenetic groups, A and B (Andries, K., J. Virol., 1990; 64:1117-1123), and new genetic variants of HRV have recently been reported. (Lamson, D., J. Infect. Dis., 2006; 194:1398-1402; McErlean, P., J. Clin. Virol., 2007; 39:67-75.)
Clinically, presentation of HRV infection is of little diagnostic value due to symptomatic similarity with numerous other infectious agents. Compounding problems with HRV identification, laboratory diagnosis suffers from the failure of some strains to grow in cell culture and by their extreme antigenic variability, precluding routine use of antigen detection methods or serology. (Lu, X., J. Clin. Microbiol. 2008; 46(2):533-9.) HRV identification with prior art methods is difficult, and distinguishing HRVs from HEVs in the same clinical sample using acid liability is ineffective for many strains. Thus, modern efforts have attempted to use reverse-transcription polymerase chain reaction (RT-PCR) assays to increase the detection sensitivity and differentiation of HRVs from co-existing infectious agents.
Nucleic acid assays for HRV typically target the 5′-noncoding region (5′NCR) of the viral genome. The 5′NCR is preferred due to the availability of highly conserved sequences that support the complex secondary structures of the HRV/HEV internal ribosome entry site (Witwer, C., Nucleic. Acids Res., 2001; 29:5079-5089). Whereas the locations of these conserved sequences offer considerable flexibility for designing targeted primer/probes for HEV real-time RT-PCR assays (Kares, S., J. Clin. Virol., 2004; 29:99-104, Nijhuis, M., J. Clin. Microbiol., 2002; 40:3666-3670; Verstrepen, W. A., J. Clin. Microbiol.; 2001; 39:4093-4096), development of comparable assays for HRVs is hampered by their greater genetic variability and the paucity of published HRV sequence data from the 5′-NCR. In addition, prior art nucleic acid assays require post-amplification processing of the amplicon by gel electrophoresis, probe hybridization, sequencing or restriction analysis to confirm and differentiate HRVs from HEVs (Andeweg, A. C., J. Clin. Microbiol., 1999; 37:524-530; Atmar, R. L., and Georghiou, J. Clin. Microbiol., 1993; 31:2544-2546; Billaud, G., J. Virol. Methods, 2003; 108:223-228; Blomqvist, S., J. Clin. Microbiol., 1999; 37:2813-2816; Halonen, P., J. Clin. Microbiol., 1995; 33:648-653; Kammerer, U., J. Clin. Microbiol., 1994; 32:285-291; Loens, K., J. Clin. Microbiol., 2006; 44:166-171; Miller, E. K., J. Infect. Dis., 2007; 195:773-781; Papadopoulos, N. G., J. Virol. Methods, 1999; 80:179-85).
More recently, real-time RT-PCR assays have been described for HRV/HEVs (Dagher, H., J. Virol. Methods., 2004; 117:113-121; Deffernez, C., J. Clin. Microbiol., 2004; 42:3212-3218; Kares, S., J. Clin. Virol., 2004; 29:99-104, Nijhuis, M., J. Clin. Microbiol., 2002; 40:3666-3670; Scheltinga, S. A., J. Clin. Virol., 2005; 33:306-311). These assays did not detect all known HRV serotypes (Dagher, H., J. Virol. Methods., 2004; 117:113-121; Deffernez, C., J. Clin. Microbiol., 2004; 42:3212-3218; Scheltinga, S. A., J. Clin. Virol., 2005; 33:306-311; Wright, P. F., J. Clin. Microbiol., 2007; 45:2126-2129) or used difficult to interpret SYBR Green detection (Dagher, H., J. Virol. Methods., 2004; 117:113-121; Wittwer, C T., Biotechniques, 1997; 22:130-131, 134-138). Moreover, these prior art assays are inaccurate due to the extensive genetic variability of the HRVs and lack of available sequence data in the public domain. Thus, no real-time RT-PCR assays specifically identify all HRVs relative to HEVs or other viral fluid components (Dagher, H., J. Virol. Methods., 2004; 117:113-121; Deffernez, C., J. Clin. Microbiol., 2004; 42:3212-3218, Scheltinga, S. A., J. Clin. Virol., 2005; 33:306-311; Wright, P. F., J. Clin. Microbiol., 2007; 45:2126-2129). Finally, no prior art assay has successfully detected viral prototype strains. Thus, there is a need for a rapid, sensitive, and discriminatory assay for detection of HRV in complex clinical or laboratory samples in the presence or absence of other viral agents.